Quick Differential Gene Analysis
Steve Pederson
22 May 2019
Setup
Data Description
- The initial files were single-end reads of 100bp
- Library sizes were between 37,710,559 and 44,890,754
- Low levels of adapter content were found and removed using
AdapterRemoval - Spikes in GC content at around 63% and 72% were noted as potentially indicative of incomplete rRNA removal
- rRNA reads were not removed as they will be ignored during summarisation
- Reads were aligned to the Rnor6.0 genome sourced from Ensembl Release 96, using
STAR 2.7.0dfor visualisation and inspection - Reads were psuedo-aligned to the transcriptome from Ensembl Release 96 for summarisation to the transcript-level using
kallisto 0.43.1.kallistois able to proportionally assign multi-mapping reads- Transcript-level counts were then summarised to the gene-level.
GC content of each library showing uneven and ‘spikey’ patterns. Peaks on the RHS indicate potential incomplete rRNA removal, whilst the other indicate possible excessive PCR cycles and high levels of duplicated sequences.
STAR Alignment summary showing very low levels of unique alignments, which is again suggestive of incomplete rRNA removal. Uniquely mapping reads are now within the range usually seen after summarising to genes
After summarisation the library sizes ranged between 6,954,273 and 14,565,667
- This is a disappointing result, but still manageable
- Low expressed genes will be aggressively removed as they will be unreliable given the small libraries
- Given the likelihood of high PCR duplicates, counts at the higher end will generally be more stable whilst those at the lower end will be less stable and removed.
Analysis
## Importance of components:
## PC1 PC2 PC3 PC4 PC5 PC6
## Standard deviation 69.5070 45.8828 28.5379 19.34833 15.64034 9.792e-14
## Proportion of Variance 0.5772 0.2515 0.0973 0.04473 0.02923 0.000e+00
## Cumulative Proportion 0.5772 0.8287 0.9261 0.97077 1.00000 1.000e+00| ID | Name | logFC | AveExpr | t | P.Value | FDR | Location |
|---|---|---|---|---|---|---|---|
| ENSRNOG00000047551 | Pnpla2 | -7.193 | 0.1297 | -13.55 | 3.224e-06 | 0.02734 | 1:213997709-214002815:(+) |
| ENSRNOG00000050004 | LOC100909761 | 6.555 | -1.417 | 13.04 | 4.155e-06 | 0.02734 | 9:37727942-37747514:(+) |
| ENSRNOG00000032832 | Mmp10 | 3.502 | 2.164 | 9.505 | 3.31e-05 | 0.1411 | 8:5734348-5742243:(+) |
| ENSRNOG00000032626 | Mmp3 | 3.614 | 4.589 | 8.851 | 5.236e-05 | 0.1411 | 8:5676665-5698579:(+) |
| ENSRNOG00000002802 | Cxcl1 | 4.896 | 1.902 | 8.303 | 7.86e-05 | 0.1411 | 14:18743685-18745457:(-) |
| ENSRNOG00000010278 | Il6 | 4.791 | 0.1518 | 8.274 | 8.035e-05 | 0.1411 | 4:3043231-3047807:(+) |
| ENSRNOG00000007545 | Angptl4 | 1.852 | 3.859 | 8.232 | 8.3e-05 | 0.1411 | 7:18627808-18634079:(-) |
| ENSRNOG00000060849 | Rcor2 | -5.115 | -1.303 | -8.129 | 8.987e-05 | 0.1411 | 1:222519615-222524779:(+) |
| ENSRNOG00000000239 | Ccl7 | 2.753 | 2.899 | 8.037 | 9.652e-05 | 0.1411 | 10:69423086-69424979:(+) |
| ENSRNOG00000024705 | Rarres2 | 2.478 | 4.591 | 7.585 | 0.0001388 | 0.1548 | 4:78205812-78208767:(-) |
Genes to an FDR of 15.0%
Genes up to an FDR of 20%
Session Info
R version 3.5.2 (2018-12-20)
**Platform:** x86_64-pc-linux-gnu (64-bit)
locale: LC_CTYPE=en_AU.UTF-8, LC_NUMERIC=C, LC_TIME=en_AU.UTF-8, LC_COLLATE=en_AU.UTF-8, LC_MONETARY=en_AU.UTF-8, LC_MESSAGES=en_AU.UTF-8, LC_PAPER=en_AU.UTF-8, LC_NAME=C, LC_ADDRESS=C, LC_TELEPHONE=C, LC_MEASUREMENT=en_AU.UTF-8 and LC_IDENTIFICATION=C
attached base packages: stats4, parallel, stats, graphics, grDevices, utils, datasets, methods and base
other attached packages: ggrepel(v.0.8.0), plyranges(v.1.2.0), rtracklayer(v.1.42.2), GenomicRanges(v.1.34.0), GenomeInfoDb(v.1.18.2), IRanges(v.2.16.0), S4Vectors(v.0.20.1), scales(v.1.0.0), ngsReports(v.0.99.7), BiocGenerics(v.0.28.0), pander(v.0.6.3), magrittr(v.1.5), forcats(v.0.4.0), stringr(v.1.4.0), dplyr(v.0.8.0.1), purrr(v.0.3.2), readr(v.1.3.1), tidyr(v.0.8.3), tibble(v.2.1.1), ggplot2(v.3.1.0), tidyverse(v.1.2.1), edgeR(v.3.24.3) and limma(v.3.38.3)
loaded via a namespace (and not attached): nlme(v.3.1-137), bitops(v.1.0-6), matrixStats(v.0.54.0), lubridate(v.1.7.4), RColorBrewer(v.1.1-2), httr(v.1.4.0), tools(v.3.5.2), backports(v.1.1.3), R6(v.2.4.0), lazyeval(v.0.2.2), colorspace(v.1.4-1), withr(v.2.1.2), tidyselect(v.0.2.5), compiler(v.3.5.2), cli(v.1.1.0), rvest(v.0.3.2), Biobase(v.2.42.0), Cairo(v.1.5-9), xml2(v.1.2.0), DelayedArray(v.0.8.0), plotly(v.4.8.0), ggdendro(v.0.1-20), labeling(v.0.3), digest(v.0.6.18), Rsamtools(v.1.34.1), rmarkdown(v.1.12), XVector(v.0.22.0), pkgconfig(v.2.0.2), htmltools(v.0.3.6), highr(v.0.8), htmlwidgets(v.1.3), rlang(v.0.3.2), readxl(v.1.3.1), rstudioapi(v.0.10), shiny(v.1.2.0), generics(v.0.0.2), zoo(v.1.8-5), hwriter(v.1.3.2), jsonlite(v.1.6), crosstalk(v.1.0.0), BiocParallel(v.1.16.6), RCurl(v.1.95-4.12), GenomeInfoDbData(v.1.2.0), Matrix(v.1.2-17), Rhdf5lib(v.1.4.3), Rcpp(v.1.0.1), munsell(v.0.5.0), stringi(v.1.4.3), yaml(v.2.2.0), MASS(v.7.3-51.1), SummarizedExperiment(v.1.12.0), zlibbioc(v.1.28.0), rhdf5(v.2.26.2), plyr(v.1.8.4), grid(v.3.5.2), promises(v.1.0.1), crayon(v.1.3.4), lattice(v.0.20-38), Biostrings(v.2.50.2), haven(v.2.1.0), hms(v.0.4.2), locfit(v.1.5-9.1), knitr(v.1.22), pillar(v.1.3.1), XML(v.3.98-1.19), glue(v.1.3.1), evaluate(v.0.13), ShortRead(v.1.40.0), latticeExtra(v.0.6-28), data.table(v.1.12.0), modelr(v.0.1.4), httpuv(v.1.5.0), cellranger(v.1.1.0), gtable(v.0.3.0), assertthat(v.0.2.1), xfun(v.0.5), mime(v.0.6), xtable(v.1.8-3), broom(v.0.5.1), later(v.0.8.0), viridisLite(v.0.3.0), truncnorm(v.1.0-8) and GenomicAlignments(v.1.18.1)